Project description:To obtain the global gene expression trends during ovule development, we collected samples of the gynoecium in developmental stages 9–10, 11, and 12 with three biological replicates to perform microarray assay.
Project description:Cotton fibers are seed trichomes, and their development undergoes a series of rapid and dynamic changes from fiber cell initiation, elongation to primary and secondary wall biosynthesis and fiber maturation. Previous studies showed that cotton homologues encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs). miRNAs are ~21 nucleotide (nt) RNA molecules derived from non-coding endogenous genes and mediate target regulation by mRNA degradation or translational repression. Here we show that among ~4-million reads of small RNAs derived from the fiber and non-fiber tissues, the 24-nt small RNAs were most abundant and were highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 28 putative miRNAs families, including 25 conserved and 3 novel miRNAs were identified in at least one of the cotton tissues examined. Thirty-two pre-miRNA hairpins representing 19 unique families were detected in Cotton Gene Indices version 9 (CGI9) using mirCheck. Sequencing, miRNA microarray, and small RNA blot analyses showed that many of these miRNAs differentially accumulated during ovule and fiber development. The cotton miRNAs examined triggered target cleavage in the same predicted sites of the cotton targets in ovules and fibers as that of the orthologous target genes in Arabidopsis. Targets of the potential new cotton miRNAs matched the previously characterized ESTs derived from cotton ovules and fibers. The miRNA targets including those encoding auxin response factors were differentially expressed during fiber development. We suggest that both conserved and new miRNAs play an important role in the rapid and dynamic process of fiber and ovule development in cotton.
Project description:Cotton ovule development, mutant vs wild type, Comparisons of DP16 0 dpa ovule Keywords: WildType vs Mutant 7 comparisons and one 0 dpa control. The 1A/DP16 & 4A/DP16 comparisons have 8 slides each, 4 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. The SL1-7-1/DP16 comparison has 6 slides, 3 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. The 5B/DP16, fl/Xu-142, OI/II+N & 53/DP16 comparisons have 4 slides each, 2 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. The 0 dpa control experiment has 3 slides , 3 Biological replicates, no technical replicates and no dye swapping.
Project description:DNA methylation is essential for plant and animal development. In plants, methylation occurs at CG, CHG, and CHH (H = A, C or T) sites via distinct pathways. Cotton is an allotetraploid consisting of two progenitor genomes. Each cotton fiber is a rapidly-elongating cell derived from the ovule epidermis, but the molecular basis for this developmental transition is unknown. Here we analyzed methylome, transcriptome, and small RNAome and revealed distinct changes in CHH methylation during ovule and fiber development. In ovules, CHH hypermethylation in promoters correlated positively with siRNAs, inducing RNA-dependent DNA methylation (RdDM), and up-regulation of ovule-preferred genes. In fibers, the ovule-derived cells generated additional heterochromatic CHH hypermethylation independent of RdDM, which repressed transposable elements (TEs) and nearby genes including fiber-related genes. Furthermore, CHG and CHH methylation in genic regions contributed to homoeolog expression bias in ovules and fibers. Inhibiting DNA methylation using 5-aza-2'-deoxycytidine in cultured ovules has reduced fiber cell number and length, suggesting a potential role for DNA methylation in fiber development. Thus, RdDM-dependent methylation in promoters and RdDM-independent methylation in TEs and nearby genes could act as a double-lock feedback mechanism to mediate gene and TE expression, potentiating the transition from epidermal to fiber cells during ovule and seed development.
Project description:DNA methylation is essential for plant and animal development. In plants, methylation occurs at CG, CHG, and CHH (H = A, C or T) sites. CHH methylation is established by the small RNA-directed DNA methylation (RdDM) pathway. Cotton is an allotetraploid consisting of two progenitor genomes, and each cotton fiber is a rapidly-elongating cell from the ovule epidermis. Here we show that inhibiting DNA methylation impairs fiber development. Genome-wide bisulfite -, mRNA-, and small RNA-sequencing analyses reveal that CHH hypermethyaltion through RdDM in euchromatin is associated with expression changes of nearby genes in ovules. The ovule-derived fiber cells not only maintain euchromatic CHH hypermethylation, but also generate additional heterochromatic CHH hypermethylation independent of RdDM. Moreover, CHG and CHH methylation in promoter and transcribed regions contribute to the expression bias of homoeologous genes in the allotetraploid cotton. This epigenetic and expression dynamics of developmental regulation could provide a molecular basis for natural selection and domestication of plants and animals.
Project description:DNA methylation is essential for plant and animal development. In plants, methylation occurs at CG, CHG, and CHH (H = A, C or T) sites. CHH methylation is established by the small RNA-directed DNA methylation (RdDM) pathway. Cotton is an allotetraploid consisting of two progenitor genomes, and each cotton fiber is a rapidly-elongating cell from the ovule epidermis. Here we show that inhibiting DNA methylation impairs fiber development. Genome-wide bisulfite -, mRNA-, and small RNA-sequencing analyses reveal that CHH hypermethyaltion through RdDM in euchromatin is associated with expression changes of nearby genes in ovules. The ovule-derived fiber cells not only maintain euchromatic CHH hypermethylation, but also generate additional heterochromatic CHH hypermethylation independent of RdDM. Moreover, CHG and CHH methylation in promoter and transcribed regions contribute to the expression bias of homoeologous genes in the allotetraploid cotton. This epigenetic and expression dynamics of developmental regulation could provide a molecular basis for natural selection and domestication of plants and animals.
Project description:DNA methylation is essential for plant and animal development. In plants, methylation occurs at CG, CHG, and CHH (H = A, C or T) sites. CHH methylation is established by the small RNA-directed DNA methylation (RdDM) pathway. Cotton is an allotetraploid consisting of two progenitor genomes, and each cotton fiber is a rapidly-elongating cell from the ovule epidermis. Here we show that inhibiting DNA methylation impairs fiber development. Genome-wide bisulfite -, mRNA-, and small RNA-sequencing analyses reveal that CHH hypermethyaltion through RdDM in euchromatin is associated with expression changes of nearby genes in ovules. The ovule-derived fiber cells not only maintain euchromatic CHH hypermethylation, but also generate additional heterochromatic CHH hypermethylation independent of RdDM. Moreover, CHG and CHH methylation in promoter and transcribed regions contribute to the expression bias of homoeologous genes in the allotetraploid cotton. This epigenetic and expression dynamics of developmental regulation could provide a molecular basis for natural selection and domestication of plants and animals.
Project description:Ovule development is a key process for plant reproduction that ensures correct seed production. Understanding the molecular mechanisms that control ovule formation will also provide new approaches to increase crop yield for breeding. Several molecular factors and plant hormones, including gibberellins, are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been proved to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA act remained unknown. Here we have proved that DELLA proteins control ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, thus GAI would function by its direct protein-protein interaction with CUC2 in cells of the placenta that determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and colocalization in placenta support this hypothesis. Moreover, molecular analysis of the loci at which GAI protein may act as transcriptional co-regulators in a CUC2-dependent manner identified a subset of target genes that would be regulated by the GAI-CUC2 complex and contribute to regulate ovule primordia emergence.
Project description:This experiment was designed to identify genes expressed preferentially in the two integuments of the Arabidopsis ovule. Pistils from wild type and two ovule mutants were compared against each aintegumenta-4 (ant-4) which lacks both integuments and inner no outer (ino-1) which lacks the outer integument. Genes that are highly expressed only in the integuments were expected to be reduced in expression in the mutants, as compared with wild type. Pistils containing ovules through all stages of ovule development prior to pollination were pooled for one experiment (FULL arrays), and for two separate experiments, a set of early differentiation stages (EARLY arrays) and a set of later differentiation stages (LATE ARRAYS) were pooled. Wild type and mutant lines are in the ecotype Landsberg erecta.